Natural killer (NK) cells are a fundamental component of innate immunity against tumors and virally infected cells. NK cell function is regulated by a dynamic balance between activating receptors that trigger NK cytolytic activity and inhibitory receptors that detect the lack of normal MHC class I (MHC-I) expression. The past few years have witnessed major advances in our understanding of NK cell function, including the discovery that Ly49 NK receptors not only interact with MHC-I on target cells (in trans), but also with MHC-I on the NK cell itself (in cis), the demonstration that mouse cytomegalovirus (MCMV) evades detection by NK cells by expressing an MHC-like decoy ligand (m157) for Ly49s, and the identification of novel, non-MHC ligands for the orphan NK receptors KLRG1, NKp80, and NKp30. We propose to establish the structural basis for each of these newly discovered NK receptor-ligand interactions using X-ray crystallography, and to correlate this information with analyses of NK cell function. Our objectives are: 1. Basis for Ly49 engagement of MHC-I in cis versus trans. By lowering the threshold for NK cell activation, cis interactions optimize discrimination between normal and diseased cells. Using the structure of a Ly49 receptor with its stalk region, we have constructed a model for cis-trans interactions that we will test at the cellular level through functional and biochemical analyses of structure-based Ly49 mutants. 2. Basis for direct recognition of a viral pathogen by Ly49 receptors. To compare the interaction of NK receptors with viral versus host ligands, we will determine structures of the MCMV protein m157 bound to Ly49I and Ly49H. 3. E-cadherin interactions with KLRG1 and integrin 1E27. The inhibitory receptor KLRG1 directs NK cytolysis against tumors that have lost E-cadherin expression. The structure of KLRG1 bound to E-cadherin suggests an oligomerization model for KLRG1-E-cadherin interactions and implies that E-cadherin can co-engage KLRG1 and 1E27. We will test the oligomerization and co-engagement hypotheses. 4. Basis for cellular cross-talk via the activating receptors NKp80 and AICL. The NKp80-AICL interaction not only promotes NK cell- mediated cytolysis of malignant myeloid cells, but is also crucial for the mutual activation of NK cells and monocytes at inflammation sites. To understand the recognition event underpinning this cross-talk, we will determine the structure of NKp80 bound to AICL. 5. Basis for recognition of BAT3, a tumor- derived nuclear factor, by the natural cytotoxicity receptor NKp30. Following its release from tumor cells, BAT3 binds to NKp30 and triggers NKp30-mediated cytotoxicity. As a nuclear factor, BAT3 defines a completely new class of NK receptor ligand. Moreover, its identification as a danger signal makes the NKp30-BAT3 interaction of special interest for understanding how the immune system recognizes damaged cells. We propose to determine the structure of BAT3 in complex with NKp30. PUBLIC HEALTH RELEVANCE: Natural killer (NK) cells play a central role in innate immune responses to a variety of virally infected or malignant cells. The goal of this proposal is to obtain structural insights into the interaction of NK cell receptors with viral or host ligands that regulate NK cell function during viral infections or tumor immunosurveillance. Such knowledge may contribute to the development of new strategies for the treatment of viral infections or cancers based on modulation of NK cell cytolytic activity.